Antibacterial mutilins

ABSTRACT

A compound of formula (I) wherein R and R 2  and the attached nitrogen atom form pyrrolidinyl or piperidinyl. R 1  is a group of formula (II). R 3  and R′ 3  are hydrogen, deuterium or halogen. R 4 , R 5  and R 10  are independently of each other hydrogen or alkyl. R 6 , R 7  and R 8  are hydrogen or deuterium. R 9  is amino, alkyl, aryl, heterocyclyl or mercapto. If X is oxygen, R 9  is additionally hydrogen; R′ 10  is alkyl, X is sulphur, oxygen, NR 10 , or N+(R′ 10 ) 2 ; Y is sulphur or oxygen, and m is 0,1 or 2, with the proviso that, when R and R 2  and the attached nitrogen atom form piperidinyl, m is O, Y is S and Y is attached in position 3 of the piperidine ring, that group of formula (I) which is attached to the piperidine ring via the residue Y is either in the (S)-configuration or in the (R)-configuration, preferably in the (S)-configuration which is useful as antimicrobial, antibacterial.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.10/363,840, filed on Mar. 10, 2003 and issued as U.S. Pat. No. 7,169,804on Jan. 30, 2007, which is a National Stage application of InternationalApplication No. PCT/EP2001/10502, filed on Sep. 11, 2001, which claimspriority of United Kingdom Application No. 0022440.2, filed on Sep. 13,2000, United Kingdom Application No. 0022439.4, filed on Sep. 13, 2000and United Kingdom Application No. 0024674.4, filed on Oct. 9, 2000.

The present invention relates to compounds having e.g. antimicrobial,such as antibacterial, activity; more specifically the present inventionrelates to mutilins.

In one aspect the present invention provides a compound of formula

wherein

R and R₂ together with the nitrogen atom to which they are attached formpyrrolidinyl or piperidinyl,

R₁ is a group of formula

R₃ and R′₃ are hydrogen, deuterium or halogen,

R₄ is hydrogen or alkyl, e.g. (C₁₋₄)alkyl,

R₅ is hydrogen or alkyl, e.g. (C₁₋₄)alkyl,

R₆, R₇ and R₈ are hydrogen or deuterium;

R₉ is amino, alkyl, aryl, heterocyclyl or mercapto; and, if X is oxygen,R₉ is additionally hydrogen;

R₁₀ is hydrogen or alkyl, e.g. (C₁₋₄)alkyl,

R′₁₀ is alkyl, e.g. (C₁₋₄)alkyl,

X is sulphur, oxygen, NR₁₀, or N⁺(R′₁₀)₂ e.g. in the presence of anappropriate anion;

Y is sulphur or oxygen, and

m is 0, 1 or 2;

with the proviso that, when R and R₂ together with the nitrogen atom towhich they are attached form piperidinyl, m is 0, Y is S and Y isattached in position 3 of said piperidine ring that group of formula Iwhich is attached to the piperidine ring via the residue Y is either inthe (S)-configuration or in the (R)-configuration, preferably in the(S)-configuration, e.g. with the proviso that, when R and R₂ togetherwith the nitrogen atom to which they are attached form piperidinyl, m is0, Y is S and Y is attached in position 3 of said piperidine ring, apart of R₉ is either in the (S)-configuration or in the(R)-configuration, e.g., if R₉ is alkyl substituted by amino, that aminegroup is either in the (S)-configuration or in the (R)-configuration;e.g. with the proviso that, if in a compound of formula I m is 0, a partof formula I is either in the (S)-configuration or in the(R)-configuration, e.g. if R₉ is alkyl substituted by amino, that aminegroup is either in the (S)-configuration or in the (R)-configuration.

An anion in a group of formula N⁺(R′₁₀)₂ includes appropriate anions,e.g. anions as conventional in an ammonium group as a counterion.

In a compound of formula I

R₃, R′₃, R₄, R₅, R₆, R₇ and R₈ are preferably hydrogen;

R and R₂ together with the nitrogen atom to which they are attached formpyrrolidine or piperidine (with the proviso as indicated above), e.g.unsubstituted pyrrolidine or piperidine (beside being substituted by agroup of formula —C(═X)R₉), or substituted pyrrolidine or piperidine(further subsituted beside being substituted by a group of formula—C(═X)R₉), e.g. substituted by one or more groups which are conventionalin organic chemistry, e.g. pleuromutilin chemistry. Preferablypyrrolidine or piperidine are unsubstituted (beside being substituted bya group of formula —C(═X)R₉)). In a compound of formula I pyrrolidineand piperidine include a group —N(R₁) and are bound to a group—(CH₂)_(m)—Y. The group —N(R₁) and the group —(CH₂)_(m)—Y may be vicinalor in another position in the pyrrolidine or piperidine ring, e.g. inpositions 1,2; 1,3; 1,4; 1,5, and, in case of piperidine, 1,6; and arepreferably in positions 1,3 or 1,4 in case of the piperidine ring and inpositions 1,2 or 1,3 in case of the pyrrolidine ring;

R₉ is preferably alkyl, e.g. (C₁₋₈)alkyl, such as (C₁₋₄)alkyl, e.g.unsubstituted or substituted alkyl, e.g. substituted by groups which areconventional in pleuromutilin chemistry, e.g. one or more amino,heterocyclyl, e.g. including a 5 or 6 membered ring containing 1 or 2nitrogen atoms; e.g. imidazolyl. If R₉ is alkyl substituted by amino,e.g. and heterocyclyl, R₉ is preferably that part of an amino acid whichremains if the carboxylic group is split off, e.g. the group —C(═X)—wherein X is oxygen can be regarded as the carbonyl part of said aminoacid;

X is preferably oxygen;

Y is preferably sulphur, and

m is preferably 0 or 1.

If not otherwise defined herein heterocyclyl includes a 5 or 6 memberedring having 1 to 4 heteroatoms selected from S, O and N; e.g. N;optionally condensed with a further ring (system), e.g. condensed with aphenyl ring; e.g. or condensed with a heterocyclyl ring. Heterocyclylincludes unsubstituted or substituted heterocyclyl, e.g. substituted bygroups which are conventional in organic chemistry, e.g. pleuromutilinchemistry. Alkyl includes (C₁₋₈)alkyl, e.g. (C₁₋₄)alkyl. Aryl includesphenyl. Amino includes a free amine group, alkyl- and dialkylamine.

In another aspect the present invention provides a compound of formulaI, wherein

R₃, R′₃, R₄, R₅, R₆, R₇ and R₈ are hydrogen;

R₁ is a group of formula

R and R₂ together with the nitrogen atom to which they are attached formpiperidine or pyrrolidine; R₉ is alkyl; X is oxygen; Y is sulphur; and mis 0 or 1;

with the proviso that, when R and R₂ together with the nitrogen atom towhich they are attached form piperidinyl, m is 0, Y is S and Y isattached in position 3 of said piperidine ring, that group of formula Iwhich is attached to the piperidine ring via the residue Y is either inthe (S)-configuration or in the (R)-configuration, preferably in the(S)-configuration;

e.g. with the proviso that, when R and R₂ together with the nitrogenatom to which they are attached form piperidinyl, m is 0, Y is S and Yis attached in position 3 of said piperidine ring, a part of R₉ iseither in the (S)-configuration or in the (R)-configuration, e.g. if R₉is alkyl substituted by amino, that amine group is either in the(S)-configuration or in the (R)-configuration;

e.g. with the proviso that, if in a compound of formula I m is 0, a partof formula I is either in the (S)-configuration or in the(R)-configuration, e.g. if R₉ is alkyl substituted by amino, that aminegroup is either in the (S)-configuration or in the (R)-configuration.

In another aspect the present invention provides a compound of formula

wherein R_(3p), R′_(3p), R_(6p), R_(7p) and R_(8p) are, index-numbercorrespondingly, as defined above for R₃, R′₃, R₆, R₇ and R₈; and R_(5p)is hydrogen or one or more substituents, preferably hydrogen; and if thegroup attached to the piperidine ring via the sulphur atom is inposition 3 of said piperidine ring and R_(5p) is hydrogen, then thegroup attached to the sulphur atom is either in the (S)-configuration orin the (R)-configuration, preferably in the (S)-configuration;

e.g. with the proviso that in the group attached to the nitrogen atom ofthe piperidine ring, the amine group is either in the (S)-configurationor in the (R)-configuration.

In another aspect the present invention provides a compound of formula

wherein R_(3q), R′_(3q), R_(6q), R_(7q) and R_(8q) are, index-numbercorrespondingly, as defined above for R₃, R′₃, R₆, R₇ and R₈; R_(5q) ishydrogen or one or more substituents, preferably hydrogen; and R_(q) isis that part of an amino acid which remains if the carboxylic group issplit off; e.g. including a compound of formula

wherein R′_(q) is as defined above for R_(q),

e.g. with the proviso that in a group R_(q), or R′_(q), respectively,the amine group of the amino acid residue is either in the(S)-configuration or in the (R)-configuration.

In another aspect the present invention provides a compound of formula

and of formula

wherein

R_(3r), R′_(3r), R_(6r), R_(7r) and R_(8r), or R_(3s), R′_(3s), R_(6s)R_(7s) and R_(8s), respectively, are, index-number correspondingly, asdefined above for R₃, R′₃, R₆, R₇ and R₈;

R_(5r), or R_(5s), respectively, is hydrogen or one or moresubstituents, preferably hydrogen; and

R_(1r) or R_(1s), respectively, is that part of an amino acid whichremains if the carboxylic group is split off; e.g. wherein in a compoundof formula I_(r) the group attached to the piperidine ring via thesulphur atom is either in the (S)-configuration or in the(R)-configuration; e.g.

wherein in a group R_(1r) or R_(1s), respectively, the amine group ofthe amino acid residue is either in the (S)-configuration or in the(R)-configuration.

In another aspect the present invention provides the compound, e.g. acompound selected from the group consisting of,

14-O—[(N-(3-Methyl-2-amino-buturyl-piperidin-3(S)-yl)sulfanyl)acetyl]mutilin,e.g. including

14-O—[(N-(3-Methyl-2(R)-amino-buturyl-piperidin-3(S)-yl)sulfanyl)acetyl]mutilin;and

14-O—[(N-(3-Methyl-2(S)-amino-buturyl-piperidin-3(S)-yl)sulfanyl)acetyl]mutilin;

14-O—[(N-(3-Methyl-2-amino-buturyl-piperidin-4-yl)sulfanyl)acetyl]mutilin,e.g. including

14-O—[(N-(3-Methyl-2(R)-amino-buturyl-piperidin-4-yl)sulfanyl)acetyl]mutilin,and

14-O—[(N-(3-Methyl-2(S)-amino-buturyl-piperidin-4-yl)sulfanyl)acetyl]mutilin;

14-O—[(N-(3-Methyl-2-amino-butyryl)-piperidin-3-yl)-methylsulfanylacetyl]-mutilin,e.g. including

14-O—[(N-(3-Methyl-2-amino-butyryl)-piperidine-3(S)-yl)-methylsulfanylacetyl]-mutilin,and

14-O—[(N-(3-Methyl-2-amino-butyryl)-piperidine-3(R)-yl)-methylsulfanylacetyl]-mutilin,such as

14-O—[(N-(3-Methyl-2(S)-amino-butyryl)-piperidine-3(S)-yl)-methylsulfanylacetyl]-mutilin,and

14-O—[(N-(3-Methyl-2(R)-amino-butyryl)-piperidine-3(S)-yl)-methylsulfanylacetyl]-mutilin;

14-O—[(N-(3-Methyl-2-amino-butyryl)-pyrrolidine-2-yl)-methylsulfanylacetyl]-mutilin,e.g. including

14-O—[(N-(3-Methyl-2-amino-butyryl)-pyrrolidine-2(R)-yl)-methylsulfanylacetyl]-mutilin,and

14-O—[(N-(3-Methyl-2-amino-butyryl)-pyrrolidine-2(S)-yl)-methylsulfanylacetyl]-mutilin,such as

14-O—[(N-(3-Methyl-2(R)-amino-butyryl)-pyrrolidine-2(R)-yl)-methylsulfanylacetyl]-mutilinand

14-O—[(N-(3-Methyl-2(S)-amino-butyryl)-pyrrolidine-2(R)-yl)-methylsulfanylacetyl]-mutilin,

14-O—[(N-(3-Methyl-2-amino-butyryl)-pyrrolidin-3-yl)sulfanylacetyl]mutilin,e.g. including

14-O—[(N-(3-Methyl-2(R)-amino-butyryl)-pyrrolidine-3-yl)-sulfanylacetyl]-mutilinand

14-O—[(N-(3-Methyl-2(S)-amino-butyryl)-pyrrolidine-3-yl)-sulfanylacetyl]-mutilin;and

14-O—[(N-histidinyl-pyrrolidin-3-yl)sulfanylacetyl]mutilin, e.g.including

4-O—[(N—(R)-histidinyl-pyrrolidin-3-yl)sulfanylacetyl]mutilin, and

4-O—[(N—(S)-histidinyl-pyrrolidin-3-yl)sulfanylacetyl]mutilin.

e.g. in free form or in the form of a salt, e.g. a salt withhydrochloric acid; such as a hydrochloride.

14-O—[(N-histidinyl-pyrrolidin-3-yl)sulfanylacetyl]mutilin is14-O—[(N-(3-(imidazol-4yl)-2-amino-propionyl-pyrrolidin-3-yl)sulfanylacetyl]mutilin.

Compounds provided by the present invention are hereinafter designatedas “compound(s) of the present invention”. A compound of the presentinvention includes a compound in any form, e.g. in free form, in theform of a salt, in the form of a solvate and in the form of a salt and asolvate. A compound of formula I includes a compound of formula I_(p),I_(q), I_(q)′, I_(r) and I_(s).

In another aspect the present invention provides a compound of formula Iin the form of a salt, or in the form of a salt and in the form of asolvate, or in the form of a solvate.

A salt of a compound of formula I includes a pharmaceutically acceptablesalt, e.g. including a metal salt or an acid addition salt. Metal saltsinclude for example alkali or earth alkali salts; acid addition saltsinclude salts of a compound of formula I with an acid, e.g. hydrogenfumaric acid, fumaric acid, naphthalin-1,5-sulphonic acid, hydrochloricacid, deuterochloric acid; preferably hydrochloric acid ordeuterochloric acid.

A compound of formula I in free form may be converted into acorresponding compound in the form of a salt; and vice versa. A compoundof formula I in free form or in the form of a salt and in the form of asolvate may be converted into a corresponding compound in free form orin the form of a salt in unsolvated form; and vice versa.

A compound of the present invention may exist in the form of isomers andisomeric mixtures thereof, e.g. optical isomers, cis trans configuratedisomers. A compound of the present invention may e.g. contain asymmetriccarbon atoms and may thus exist in the form of diastereoisomeres andmixtures thereof, e.g. epimers. For example in a compound of formula I,that group bound via group —(CH₂)_(m)—Y to the pyrrolidine or piperidinering may be in the (R)- and in the (S)-configuration, e.g. includingmixtures thereof. E.g. if R₉ is alkyl substituted by amine, e.g. R₉ isthat part of an amino acid which remains if the carboxylic group issplit off, said amine group in R₉ may be in the (S)- or in the(R)-configuration, e.g. including mixtures thereof.

Isomeric, diastereoisomeric and epimeric mixtures may be separated asappropriate, e.g. according to a method as conventional, to obtain pureisomers. Pure isomers may also be produced as appropriate, e.g.according, e.g. analogously, to a method as conventional, e.g. or asdescribed herein.

The present invention includes a compound of the present invention inany isomeric form and in any isomeric mixture, with the proviso that,when R and R₂ together with the nitrogen atom to which they are attachedform piperidinyl, m is 0, Y is S and Y is attached in position 3 of saidpiperidine ring, that group of formula I which is attached to thepiperidine ring via the residue Y is either in the (S)-configuration orin the (R)-configuration, preferably in the (S)-configuration;

e.g. with the proviso that, when R and R₂ together with the nitrogenatom to which they are attached form piperidinyl, m is 0, Y is S and Yis attached in position 3 of said piperidine ring, a part of R₉ iseither in the (S)-configuration or in the (R)-configuration, e.g. if R₉is alkyl substituted by amino, that amine group is either in the(S)-configuration or in the (R)-configuration;

e.g. with the proviso that, if in a compound of formula I m is 0, a partof formula I is either in the (S)-configuration or in the(R)-configuration, e.g. if R₉ is alkyl substituted by amino, that aminegroup is either in the (S)-configuration or in the (R)-configuration.

Preferably the configuration in the mutilin ring of a compound offormula I is the same as in a naturally produced mutilin.

A compound of the present invention may be obtained as appropriate, e.g.according, e.g. analogously, to a method as conventional, or, asdescribed herein.

In another aspect the present invention provides a process for theproduction of a compound of formula I as defined above comprising thesteps

a. reacting a compound of formula

wherein R₃, R′₃, R₄ and R₅ are as defined above and R₆, R₇ and R₈ arehydrogen, with urea or thiourea and subsequent reduction to obtain acompound of formula

wherein Y is as defined above; R₃, R′₃, R₄ and R₅ are as defined aboveand R₆, R₇ and R₈ are hydrogen,

b. reacting a compound of formula III as defined in step a. withoptionally substituted pyrrolidine, methyl or ethyl pyrrolidine,piperidine, methyl or ethyl piperidine (=methyl-, ethyl-pyrrolidine orpiperidine), respectively, carrying at the nitrogen atom a group offormula —C(═X)R₉, wherein X and R₉ are as defined above, in the form ofa reactive derivative, e.g. in the form of a mesylate or a tosylate; toobtain a compound of formula

which is a compound of formula I wherein R, R₁, R₂, R₃, R′₃, R₄, R₅, Yand m are as defined above and R₆, R₇ and R₈ are hydrogen; and, ifdesired,

c. introducing deuterium into a compound of formula IV as defined instep b, to obtain a compound of formula I, wherein R, R₁, R₂, R₃, R′₃,R₄, R₅, Y and m are as defined above and R₆, R₇ and R₈ are deuterium.

Groups may be unprotected or protected and may be deprotected in anystep, if desired, e.g. according, e.g. analogously to a method asconventional.

E.g. in an optionally substituted pyrrolidine, methyl or ethylpyrrolidine, piperidine, methyl or ethyl piperidine (=methyl-,ethyl-pyrrolidine or piperidine), respectively, carrying at the nitrogenatom a group of formula —C(═X)R₉, wherein R₉ is alkyl substituted byamine, said amine group may be protected or unprotected. Appropriateprotection groups include e.g. protection groups as conventional, suchas tert.butoxycarbonyl (BOC).

If in step b. as defined above the methane- or toluenesulphonic acidresidue, respectively, attached to the methyl-, ethyl-pyrrolidine orpiperidine ring is in the (R)-configuration a compound of formula Wobtained may be in a form, wherein the group attached to the methyl-,ethyl-pyrrolidine or piperidine ring via the sulphur atom is in the(S)-configuration; If in step b. as defined above the methane- ortoluenesulphonic acid residue, respectively, attached to the methyl-,ethyl-pyrrolidine or piperidine ring is in the (S)-configuration acompound of formula IV obtained may be in a form, wherein the groupattached to the methyl-, ethyl-pyrrolidine or piperidine ring via thesulphur atom is in the (R)-configuration (Walden inversion).

If in step b. as defined above in a group of formula —C(═X)R₉ attachedto the methyl-, ethyl-pyrrolidine or piperidine ring used for reactionwith a compound of formula III R₉ is alkyl substituted by amine, e.g.that part of an amino acid which remains if the carboxylic group issplit off, wherein said amine is in the (R)-configuration, a compound offormula IV is obtained, wherein said amine is in the (R)-configuration.If in step b. as defined above in a group of formula —C(═X)R₉ attachedto the methyl-, ethyl-pyrrolidine or piperidine ring used for reactionwith a compound of formula III R₉ is alkyl substituted by amine, e.g.that part of an amino acid which remains if the carboxylic group issplit off, wherein said amine is in the (S)-configuration, a compound offormula IV is obtained, wherein said amine is in the (S)-configuration.

The production of a compound of formula I, wherein R₃ and R′₃ aredeuterium or halogen may be e.g. carried out via treatment of a compoundof formula

wherein the carbon atoms carrying R₃ and R′₃, which both are hydrogen,together form a double bond with deuterium or halogen, e.g. F₂, Cl₂,Br₂, to obtain a compound of formula V, wherein R₃ and R′₃ are deuteriumor halogen; and further reacting a compound of formula V as appropriateto obtain a compound of formula I wherein R₃ and R′₃ are deuterium orhalogen.

Preferably a compound of formula II may be obtained from a compound offormula V by reacting a compound of formula V with a compound of formula

wherein Y, R₄ and R₅ are as defined above and Hal is halogen, preferablybromo, chloro. Introduction of deuterium in a compound of formula I maybe carried out by treatment of a compound of formula I, wherein R₆, R₇and R₈ are hydrogen, e.g. in the form of a hydrochloride, withdeuterochloric acid (DCl) in appropriate solvent (system); and isolationof a compound of formula I wherein R₆, R₇ and R₈ and are deuterium. If acompound of formula I is in the form of an acid addition salt, such as ahydrochloride, treatment of said salt with DCl may also result in thereplacement of the hydrogen of said acid, e.g. in a compound of formulaI in the form of a deuterochloride.

Any compound described herein, e.g. compounds of formula II, III, IV, V,VI and optionally substituted pyrrolidines, methyl or ethylpyrrolidines, piperidines, methyl or ethyl piperidines, respectively,are known or may be obtained according, e.g. analogously, to a method asconventional, e.g. or as described herein.

The compounds provided by the present invention including compounds offormula I, hereinafter designated as “active compound(s) of the presentinvention” exhibit pharmacological activity and are therefore useful aspharmaceuticals. For example, the active compounds of the presentinvention, e.g. compounds of the examples, show antimicrobial, e.g.antibacterial, activity against gram positive bacteria, such asStaphylococci, e.g. Staphylococcus aureus, Streptococci, e.g.Streptococcus pyogenes, Streptococcus pneumoniae, Enterococci, e.g.Enterococcus faecium, as well as against Mycoplasms, Chlamydia andobligatory anaerobes, e.g. Bacteroides fragilis; in vitro in the AgarDilution Test or Microdilution Test according to National Committee forClinical Laboratory Standards (NCCLS) 1997, Document M7-A4 Vol. 17, No.2: “Methods for dilution Antimicrobial Susceptibility Tests for Bacteriathat Grow Aerobically—Fourth Edition, Approved Standard”; and in theAnaerobic Bacteria TEST according to National Committee for ClinicalLaboratory Standards (NCCLS) VOL. 13, No. 26, M11-A4, Methods forAntimicrobial Susceptibility Testing of Anaerobic Bacteria; ApprovedStandard; Fourth Edition (1997).

The active compounds of the present invention show antibacterialactivity in vitro (MIC (μg/ml)) in the Agar Dilution Test or in theMicrodilution Test from about ≦0.01 μg/ml to 25 μg/ml, e.g. againstabove mentioned bacterial species; and are active against Mycoplasms andChlamydia. MIC=minimal inhibitory concentration.

The active compounds of the present invention show e.g. activity insystemic infections of mice determined according to the Handbook ofanimal models of infection. Edition Oto Zak and Merle A. Sande, AcademicPress, 1999), e.g. against Staphylococcus, e.g. when administeredparenterally or orally, e.g. at dosages from about 5 to 150 mg/kg bodyweight; e.g. the ED₅₀ values for the compounds14-O—[(N—(R)-histidinyl-pyrrolidin-3 -yl)sulfanylacetyl]mutilin or14-O—[(N-(3-methyl-2(R)-amino-butyryl)-pyrrolidine-3-yl)-sulfanylacetyl]-mutilinis of about 11.0 mg/kg body weight; and the ED₅₀ values for the compound14-O—[(N-(3-methyl-2(R)-amino-buturyl)-piperidin-3(S)-yl)sulfanyl)acetyl]-mutilin,is in the range of 7.0 mg/kg body weight.

ED₅₀=Effective dosage in mg/kg body weight per application by which 50%of the treated animals are protected from death; calculated by Probitanalysis (n=8 animals/group).

In another aspect the present invention provides a compound of thepresent invention for use as a pharmaceutical, preferably as anantimicrobial, such as an antibiotic, e.g. and as an anti-anaerobic.

In another aspect the present invention provides a compound of thepresent invention for use in the preparation of a medicament for thetreatment of microbial diseases, for example of diseases caused bybacteria, e.g. selected from Staphylococci, Streptococci, Enterococci;e.g. and of diseases caused by Mycoplasms, Chlamydia and obligatoryanaerobes.

Surprisingly the active compounds of the present invention show alsoactivity against strains which are resistant against erythromycin(s),tetracycline(s), e.g. strains including penicillin ormultidrug-resistant strains, e.g. of Staphylococcus aureus (MRSA).

In another aspect the present invention provides a method of prophylaxisand treatment of microbial diseases which comprises administering to asubject in need of such treatment an effective amount of a compound ofthe present invention, e.g. in the form of a pharmaceutical composition.

For prophylaxis and treatment of microbial diseases, the appropriatedosage will, of course, vary depending upon, for example, the activecompound of the present invention employed, the host, the mode ofadministration and the nature and severity of the conditions beingtreated. However, in general, for satisfactory results in largermammals, for example humans, an indicated daily dosage is in the rangefrom about 0.5 to 3 g, of an active compound of the present inventionconveniently administered, for example, in divided doses up to fourtimes a day.

An active compound of the present invention may be administered by anyconventional route, preferably orally, e.g. in form of tablets, powders,capsules, suspensions; e.g. including non-resorbable oral formulations;or parenterally, e.g. in the form of injectable solutions orsuspensions; or topically, e.g. in the form of nasal sprays, bodysolutions, creams, eye drops. The active compounds of the presentinvention may be administered in analogous manner, e.g. in similar dosesand for similar indications, as erythromycin(s), tetracycline(s).

The active compounds of the present invention may be administered in theform of a pharmaceutically acceptable salt, e.g. an acid addition saltor metal salt;

or in free form; optionally in the form of a solvate. The activecompounds of the present invention in the form of a salt exhibit thesame order of activity as the active compounds of the present inventionin free form.

In another aspect the present invention provides a pharmaceuticalcomposition comprising a compound of the present invention in free formor in the form of a pharmaceutically acceptable salt; e.g. and/or in theform of a solvate; in association with at least one pharmaceuticalcarrier or diluent.

Such compositions may be manufactured according, e.g. analogously, to amethod as conventional. Unit dosage form may contain, for example, about100 mg to about 1 g.

The active compounds of the present invention are additionally suitableas veterinary agents, e.g. veterinary active compounds, e.g. in theprophylaxis and in the treatment of microbial, e.g. bacterial diseases,in animals, such as fowl, pigs and calves; e.g. and for diluting fluidsfor artificial insemination and for egg-dipping techniques.

In another aspect the present invention provides a compound of thepresent invention for use as a veterinary agent.

In another aspect the present invention provides a compound of thepresent invention for the preparation of a veterinary composition whichis useful as a veterinary agent e.g. in the prophylaxis and in thetreatment of microbial, e.g. bacterial diseases, in animals.

The present invention further provides a veterinary method for theprophylaxis and the treatment of microbial, e.g. bacterial, diseaseswhich comprises administering to a subject in need of such treatment aneffective amount of a compound of the present invention, e.g. in theform of a veterinary composition.

For use of the active compounds of the present invention as a veterinaryagent, the dosage will of course vary depending upon the size and age ofthe animal and the effect desired; for example for prophylactictreatment relatively low doses would be administered over a longer timeperiod, e.g. 1 to 3 weeks. Preferred doses in drinking water are from0.0125 to 0.05 weight by volume, particularly 0.0125 to 0.025; and infoodstuffs from 20 to 400 g/metric ton, preferably 20 to 200 g/metricton.

It is preferred to administer the active compounds of the presentinvention as a veterinary agent to hens in drinking water, to pigs infoodstuff and to calves orally or parenterally, e.g. in the form of oralor parenteral preparations.

In the following examples which illustrate the invention temperaturesare in degree Celsius and are uncorrected.

The following abbreviations are used:

BOC: tert.butoxycarbonyl

TFA: trifluoroacetic acid

DCC: dicyclohexylcarbodiimide

The numbering of the mutilin cyclus referred to in the examples is givenin the following formula:

EXAMPLES Example 114-O—[(N-(3-Methyl-2-amino-buturyl-piperidin-3-yl)sulfanyl)acetyl]mutilin

A) 14-O-[(Carbamidoylsulfanyl)acetyl]mutilin-tosylate

A solution of 15.2 g of thiourea and 106.4 g ofpleuromutilin-22-O-tosylate in 250 ml of acetone is heated under reflux,solvent is removed under reduced pressure and 100 ml of hexane areadded. A precipitate forms, is filtrated off and dried.

14-O-[(Carbamidoylsulfanyl)acetyl]mutilin-tosylate is obtained.

B) 14-Mercapto-acetyl-mutilin

A solution of 4.7 g of sodium pyrosulfite (Na₂S₂O₅) in 25 ml of H₂O isadded to a solution of 12.2 g of14-O-[(carbamimidoylsulfanyl)acetyl]mutilin-tosylate in a mixture of 20ml of ethanol and 35 ml of H₂O (warmed to ca. 90°). 100 ml of CCl₄ areadded to the reaction mixture obtained and the mixture is heated underreflux. The two-phase system obtained is separated, the organic phase isdried and the solvent is evaporated off.

14-Mercapto-acetyl-mutilin is obtained.

C) N—BOC-3(R)-Hydroxy-piperidine

A suspension of 3.48 g of 3-(R)-hydroxypiperidine, 8.72 g ofdi-tert.butyl-dicarbonat and 4.0 g of N-metyl-morpholine in 70 ml ofdioxane is stirred at room temperature. From the mixture obtained thesolvent is evaporated off and the evaporation residue is dissolved inCH₂Cl₂ and extracted with 1N HCl. The organic phase is dried and thesolvent is evaporated off.

5.08 g of N—BOC-3(R)-Hydroxy-piperidine are obtained which can be usedwithout further puification for further reaction.

D) N—BOC-3(R)-methylsulfonyloxy-piperidine

A solution of 5.08 g of N—BOC-3(R)-Hydroxy-piperidine and 8.7 g ofmethanesulfonic acid anhydride in 100 ml of pyridine is stirred at roomtemperature. Pyridine is distilled off under high vacuum and thedistillation residue obtained is dissolved in CH₂Cl₂, which is extractedwith 1N HCl. The organic phase obtained is dried and the solvent isevaporated off to dryness. The residue is purified by chromatography.

3.8 g of N—BOC-3(R)-methylsulfonyloxy-piperidine are obtained.

¹HNMR (CDCl₃): 4.7(m, 1H, CHOSO₂CH₃), 3.2-3.6 (m, 4H, CHN), 3.0 (s, 3H,CH₃SO₂), 1.4 (m, 9H, tert.butyl).

E) 14-O—[(N—BOC-Piperidin-3(S)-yl)-sulfanylacetyl]-mutilin

A solution of 1.97 g of 22-mercapto-acetyl-pleuromutilin, 1.39 g ofN—BOC-3(R)-methyl-sulfonyloxy-piperidine and 0.12 g of sodium in 50 mlof EtOH is heated to 90°. The solvent of the mixture obtained isevaporated off to dryness under vacuum and the residue is subjected tochromatography. 2.5 g of14-O—[(N—BOC-Piperidin-3(S)-yl)-sulfanylacetyl]-mutilin are obtained.

¹HNMR (CDCl₃): 6.45, 5.35, 5.2 (3×m, H₁₉, H₂₀, H₂₁), 5.74 (d, 1H, 5.2Hz, H₁₄), 3.35 (d, 1H, H₁₁, J=5.2 Hz), AB-system: 3.12,3.18, (J=14.7 Hz,H₂₂), 3.2, 2.95, 2.65,2.6 (4×m, CH₂NCH₂), 2.85 (m, 1H, SCH), 1.18, 1.45(2×s, (CH3)₁₅, CH3)₁₈), 0.75,0.88 (2×d, (CH₃)₁₈ (CH₃)₁₇, J=5.4 Hz)

F)14-O—[(N-(3-Methyl-2(R)-amino-buturyl)-piperidin-3(S)-yl)sulfanyl)acetyl]mutilinin the form of a hydrochloride

A solution of 280 mg of14-O—[(N—BOC-Piperidin-3(S)-yl)-sulfanylacetyl]-mutilin in 20 mlmethylenchloride and 1 ml TFA is stirred at room temperature for ca. 30minutes and the solvent of the mixture obtained is evaporated off todryness. To the evaporation residue obtained, dissolved in 40 ml ofCH₂Cl₂, 55 mg of N-metylmorpholine, 110 mg of N—BOC—(R)-valine and 105mg of DCC are added. The mixture obtained is stirred and precipitateddicyclohexylurea is filtrated off. The solvent from the filtrateobtained is evaporated off and the evaporation residue is subjected tochromatography.14-O—[(N-(3-Methyl-2(R)-(N—BOC-amino)-buturyl)-piperidin-3(S)-yl)sulfanyl)acetyl]mutilinis obtained and is treated with TFA. The solvent of the mixture obtainedis evaporated off to dryness and the evaporation residue is treated withetheric HCl. Solid, amorphous14-O—[(N-(3-methyl-2(R)-amino-buturyl)-piperidin-3(S)-yl)sulfanyl)acetyl]mutilinin the form of a hydrochloride is obtained.

¹HMR (d₆-DMSO, 330K): 6.45, 5.35, 5.2 (3×m, H₁₉, H₂₀, H₂₁), 5.74 (d, 1H,5.2 Hz, H₁₄), 5.45 (d, 1H, NH, J=7.8 Hz), 4.1 (m , 1H, NHCHCO), 3.35 (d,1H, H₁₁, J=5.2 Hz), AB-system: 3.12, 3.18, (J=14.7 Hz, H₂₂), 3.2, 2.95,2.65, 2.6 (4×m, CH₂NCH₂), 2.8 (m, 1H, SCH), 1.18, 1.45 (2×s, (CH3)₁₅,(CH3)₁₈), 0.75, 0.88 (2×d, (CH₃)₁₆, (CH₃)₁₇, J=5.4 Hz), 0.78, 0.84 (2×d,(CH₃)₂CH J=6.8 Hz)

Example 214-O—[(N-(3-Methyl-2-amino-buturyl-piperidin-4-yl)sulfanyl)acetyl]mutilin

According to the method described in Example 1 but using the appropriatestarting materials (e.g. 4-hydroxymethyl-piperidine instead of3-hydroxymethyl-piperidine),4-O—[(N-(3-Methyl-2(R)-amino-buturyl-piperidin-4-yl)sulfanyl)acetyl]mutilinin the form of a hydrochloride is obtained.

¹HMR (d₆-DMSO): 8.1 (b, 3H, NH3+), 6.2-6.4 (m, 1H, H19), 5.55 (d, 1H,H14), 5.1-5.2 (m, 2H, H20), 4.25 (m, 1H, NCHCO), 4.1,4.25 (m, 1H, NCH),3.8-3.95 (m, 1H, NCH), 3.4 (d, 1H, H11), 3.0-3.2 (m, 2H, NCH), 2.8-2.95(m, 1H, sCH), 2.4 (m1H, H4), 3.25-3.4 (m, 2H, SCH2CO), 1.08, 1.39 (2×s,(CH3)15, (CH3)18), 0.93,0.98 (2×d, 6H, (CH3)2CH), 0.65,0.85 (2×d, 6H,(CH3)16, (CH3)16, (CH3)17).

Example 314-O—[(N-(3-Methyl-2-amino-butyryl)-piperidin-3-yl)-methylsulfanylacetyl]-mutilin

A) N—BOC-3(R)-Hydroxymethyl-piperidine

A suspension of 3.48 g of 3-(R)-hydroxymethyl-piperidine, 8.72 g ofdi-tert.butyl-dicarbonat and 4.0 g of N-methyl-morpholine in 70 ml ofdioxane is stirred for ca. 18 hours at room temperature. From themixture obtained the solvent is evaporated off and the evaporationresidue is dissolved in CH₂Cl₂ and extracted with 1N HCl. The organicphase is dried and the solvent is evaporated off. 5.08 g ofN—BOC-3(R)-hydroxymethyl-piperidine are obtained which may be used forfurther reaction without further puification.

B) N—BOC-3(R)-Methylsulfonyloxymethyl-piperidine

A solution of 5.08 g of N—BOC-3(R)-hydroxymethyl-piperidine and 8.7 g ofmethanesulfonic acid anhydride in 100 ml of pyridine is stirred at roomtemperature for ca. 22 hours. From the mixture obtained pyridine isevaporated off (high vacuum) and the evaporation residue obtained isdissolved in CH₂Cl₂. The organic phase obtained is extracted with 1NHCl, dried and the solvent is evaporated off. The evaporation residue issubjected to chromatography. 3.8 g ofN—BOC-3(R)-methylsulfonyloxymethyl-piperidine are obtained.

C) 14-O—[(N—BOC-Piperidin-3(S)-yl)-methylsulfanylacetyl]-mutilin

A solution of 1.97 g of 22-mercapto-pleuromutilin, 1.39 g ofN—BOC-3(R)-methylsulfonyloxymethyl-piperidine and 0.12 g of sodium in 50ml of EtOH is heated at ca. 90°. The solvent of the mixture obtained isevaporated off and the evaporation residue obtained is subjected tochromatography. 2.5 g of14-O—[(N—BOC-Piperidin-3(S)-yl)-methylsulfanylacetyl]-mutilin areobtained.

D)14-O—[(N-(3-Methyl-2(S)-amino-butyryl)-piperidine-3(S)-yl)-methylsulfanylacetyl]-mutilinin the form of a hydrochloride

A solution of 280 mg of14-O—[(N—BOC-piperidin-3(S)-yl)-methylsulfanylacetyl]-mutilin in 20 mlof CH₂Cl₂ and 1 ml of TFA is stirred at room temperature for ca. 30minutes and the solvent of the mixture obtained is evaporated off. Theevaporation residue obtained is dissolved in 40 ml of CH₂Cl₂ and to thesolution obtained 55 mg of N-metylmorpholine, 110 mg of N—BOC—(S)-valineand 105 mg of DCC are added and the mixture obtained is stirred. Fromthe mixture obtained a solid (precipitated dicyclohexylurea) isfiltrated off and the filtrate obtained is subjected to chromatography.14-O—[(N—BOC-(3-Methyl-2(S)-amino-butyryl)-piperidine-3(S)-yl)-methylsulfanylacetyl]-mutilinis obtained which is treated with TFA and etheric HCl.14-O—[(N-(3-Methyl-2(S)-amino-butyryl)-piperidine-3(S)-yl)-methylsulfanylacetyl]-mutilinin the form of a hydrochloride is obtained.

¹HNMR (d₆-DMSO): 6.1-6.2 (m, 1H, H19), 5.58 (d, 1H, H14). 5.5-5.12 (m,2H, H20), 4.2 (m, 2H, NCHCO, NCH), 3.75 (m, 1H, NCH), 3.42 (d, 1H, H11),3.28-3.35 (m, 2H, SCH₂CO), 3.1 (m, 2H, SCH₂), 1.08,1.36 (2×s, 6H(CH₃)₁₅, (CH₃)₁₈), 0.95,0.98 (2×d, 6H, (CH₃)₂CH), 0.65,0.83 (2×d, 6H(CH₃)₁₆, (CH₃)₁₇.

Example 414-O—[(N-(3-methyl-2-amino-butyryl)-pyrrolidine-2-yl)-methylsulfanylacetyl]-mutilin

According to the method described in Example 3 but using the appropriatestarting materials (e.g. 2-(S)-hydroxymethyl-pyrrolidine instead of3-(R)-hydroxymethyl-piperidine and N—BOC—(R)-valine instead ofN—BOC—(S)-valine);14-O—[(N-(3-methyl-2(R)-amino-butyryl)-pyrrolidine-2(R)-yl)-methylsulfanylacetyl]-mutilinin the form of a hydrochloride is obtained.

¹HNMR (d₆-DMSO): Rotamere, 8.1 (b, 3H, NH3), 6.1-6.2 (m, 1H, H19), 5.52(d, 1H, H14), 5.5-5.12 (m, 2H, H20), 4.15 (m, 1H, NCHCO), 3.9 (m, 1H,NCH), 3.6 (m, 1H, NCH), 3.42 (d, 1H, H11), 3.28-3.35 (m, 2H, SCH₂CO),2.68,2.85 (2×dd, 2H, CHCH₂S), 1.08,1.36 (2×s, 6H (CH₃)₁₈), 0.95,0.98(2×d, 6H, (CH₃)₂CH), 0.65,0.83 (2×d, 6H (CH₃)₁₆, (CH₃)₁₇.

Example 514-O—[(N-(3-Methyl-2-amino-butyryl)-pyrrolidin-3-yl)sulfanylacetyl]mutilin

According to the method described in Example 1 but using appropriatestarting materials, e.g. N—BOC-3(R)-methylsulfonyloxy-pyrrolidineinstead of N—BOC-3(R)-methylsulfonyloxy-piperidine,14-O—[(N-(3-methyl-2(R)-amino-butyryl)-pyrrolidine-3-yl)-sulfanylacetyl]-mutilinin the form of a hydrochloride is obtained.

¹HNMR (d₆-DMSO, 330K): 6.45,5.35,5.2 (3×m,H₁₉,H₂₀,H₂₁), 5.64 (d, 1H, 5.2Hz,H₁₄), 6.3, (b, 1H, NH), 3.95 (m, 1H, NHCHCO), 3.35 (d, 1H, H₁₁, J=5.2Hz), AB-system: 3.0, 3.1, (J=14.7 Hz,H₂₂), 3.2, 2.95, 2.65, 2.6(4×m,CH₂NCH₂), 2.8 (m, 1H, SCH), 1.18, 1.45, (2×s, (CH3)₁₅, (CH3)₁₈),0.75,0.88 (2×d, (CH₃)₁₆, (CH₃)₁₇, J=5.4 Hz), 0.86,0.84 (2×d, (CH₃)₂CH,J=6.8 Hz).

Example 6 14-O—[(N-histidinyl-pyrrolidin-3-yl)sulfanylacetyl]mutilin

According to the method described in Example 1 but using appropriatestarting materials, e.g. N,N′—BOC—(R)-histidine instead ofN—BOC—(R)-valin,4-O—[(N—(R)-histidinyl-pyrrolidin-3-yl)sulfanylacetyl]mutilin in theform of a hydrochloride is obtained.

¹HNMR (d₆-DMSO, 330K): 7.45,6.73 (2×s,histidine), 6.25,5.1,5.2(3×m,H₁₉,H₂₀,H₂₁), 5.59 (d, 1H, 5.2 Hz,H₁₄), 5.45 (d, 1H, NH, J=7.8 Hz),4.5 (d, 1H, NHCHCO, J=4.5 Hz), 3.6,3.4 (2×m, 2H, NCH₂CH2), 3.35 (d, 1H,H₁₁, J=5.2 Hz), 3.4 (m, 2H, HisCH₂), ABX-system: 3.12,3.68, J=14.1 Hz,6.5 Hz,NCH₂CHS), 3.2,2.95,2.65,2.6 (4×m,CH₂NCH₂), 2.7 (m, 1H, SCH),1.18,1.45 (2×s, (CH3)₁₅, (CH3)18), 0.75,0.88 (2×d, (CH₃)₁₆, (CH₃)₁₇,J=5.4 Hz).

1. A compound of formula I_(r)

wherein R_(3r) and R′_(3r) are hydrogen, deuterium, or halogen; R_(5r)is hydrogen; R_(6r), R_(7r) and R_(8r) are hydrogen or deuterium; andR_(1r) is that part of an amino acid which remains if the carboxylicgroup is split off, or a salt thereof.
 2. A compound of claim 1 in theform of a salt.
 3. A pharmaceutical composition comprising an effectiveamount of a compound of claim 1 or a salt thereof and at least onepharmaceutically acceptable carrier or diluent therefor.
 4. Apharmaceutical composition of claim 3, wherein a compound of formulaI_(r)

wherein R_(3r) and R′_(3r) are hydrogen, deuteriurn, or halogen; R_(5r)is hydrogen; R_(6r),R_(7r) and R_(8r) are hydrogen or deuterium; andR_(1r) is that part of an amino acid which remains if the carboxylicgroup is split off is in the form of a pharmaceutically acceptable salt.